This invention relates to variation in genes that encode pharmaceutically-important proteins. In particular, this invention provides genetic variants of the human angiotensin receptor 1 (AGTR1) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
Current methods for identifying pharmaceuticals to treat disease often start by identifying, cloning, and expressing an important target protein related to the disease. A determination of whether an agonist or antagonist is needed to produce an effect that may benefit a patient with the disease is then made. Then, vast numbers of compounds are screened against the target protein to find new potential drugs. The desired outcome of this process is a lead compound that is specific for the target, thereby reducing the incidence of the undesired side effects usually caused by activity at non-intended targets. The lead compound identified in this screening process then undergoes further in vitro and in vivo testing to determine its absorption, disposition, metabolism and toxicological profiles. Typically, this testing involves use of cell lines and animal models with limited, if any, genetic diversity.
What this approach fails to consider, however, is that natural genetic variability exists between individuals in any and every population with respect to pharmaceutically-important proteins, including the protein targets of candidate drugs, the enzymes that metabolize these drugs and the proteins whose activity is modulated by such drug targets. Subtle alteration(s) in the primary nucleotide sequence of a gene encoding a pharmaceutically-important protein may be manifested as significant variation in expression, structure and/or function of the protein. Such alterations may explain the relatively high degree of uncertainty inherent in the treatment of individuals with a drug whose design is based upon a single representative example of the target or enzyme(s) involved in metabolizing the drug. For example, it is well-established that some drugs frequently have lower efficacy in some individuals than others, which means such individuals and their physicians must weigh the possible benefit of a larger dosage against a greater risk of side effects. Also, there is significant variation in how well people metabolize drugs and other exogenous chemicals, resulting in substantial interindividual variation in the toxicity and/or efficacy of such exogenous substances (Evans et al., 1999, Science 286:487-491). This variability in efficacy or toxicity of a drug in genetically-diverse patients makes many drugs ineffective or even dangerous in certain groups of the population, leading to the failure of such drugs in clinical trials or their early withdrawal from the market even though they could be highly beneficial for other groups in the population. This problem significantly increases the time and cost of drug discovery and development, which is a matter of great public concern.
It is well-recognized by pharmaceutical scientists that considering the impact of the genetic variability of pharmaceutically-important proteins in the early phases of drug discovery and development is likely to reduce the failure rate of candidate and approved drugs (Marshall A 1997 Nature Biotech 15:1249-52; Kleyn P W et al. 1998 Science 281: 1820-21; Kola I 1999 Curr Opin Biotech 10:589-92; Hill A V S et al. 1999 in Evolution in Health and Disease Stearns S S (Ed.) Oxford University Press, New York, pp 62-76; Meyer U. A. 1999 in Evolution in Health and Disease Stearns S S (Ed.) Oxford University Press, New York, pp 41-49; Kalow W et al. 1999 Clin. Pharm. Therap. 66:445-7; Marshall, E 1999 Science 284:406-7; Judson R et al. 2000 Pharmacogenomics 1:1-12; Roses AD 2000 Nature 405:857-65). However, in practice this has been difficult to do, in large part because of the time and cost required for discovering the amount of genetic variation that exists in the population (Chakravarti A 1998 Nature Genet 19:216-7; Wang D G et al 1998 Science 280:1077-82; Chakravarti A 1999 Nat Genet 21:56-60 (suppl); Stephens J C 1999 Mol. Diagnosis 4:309-317; Kwok P Y and Gu S 1999 Mol. Med. Today 5:538-43; Davidson S 2000 Nature Biotech 18:1134-5).
The standard for measuring genetic variation among individuals is the haplotype, which is the ordered combination of polymorphisms in the sequence of each form of a gene that exists in the population. Because haplotypes represent the variation across each form of a gene, they provide a more accurate and reliable measurement of genetic variation than individual polymorphisms. For example, while specific variations in gene sequences have been associated with a particular phenotype such as disease susceptibility (Roses A D supra; Ulbrecht M et al. 2000 Am J Respir Crit Care Med 161: 469-74) and drug response (Wolfe C R et al. 2000 BMJ 320:987-90; Dahl BS 1997 Acta Psychiatr Scand 96 (Suppl 391): 14-21), in many other cases an individual polymorphism may be found in a variety of genomic backgrounds, i.e., different haplotypes, and therefore shows no definitive coupling between the polymorphism and the causative site for the phenotype (Clark A G et al. 1998 Am J Hum Genet 63:595-612; Ulbrecht M et al. 2000 supra; Drysdale et al. 2000 PNAS 97:10483-10488). Thus, there is an unmet need in the pharmaceutical industry for information on what haplotypes exist in the population for pharmaceutically-important genes. Such haplotype information would be useful in improving the efficiency and output of several steps in the drug discovery and development process, including target validation, identifying lead compounds, and early phase clinical trials (Marshall et al., supra).
One pharmaceutically-important gene for the treatment of hypertension is the angiotensin receptor 1 (AGTR1) gene or its encoded product. AGTR1 is a G protein-coupled receptor that binds to the vasopressor angiotensin II, which is an important effector controlling blood pressure and volume in the cardiovascular system. AGTR1 appears to mediate the major cardiovascular effects of angiotensin II, and this is accomplished through activation of a phosphatidylinositol-calcium second messenger system (Murphy et al., Nature 1991; 351:233-236).
Pharmacologic agents that antagonize AGTR1 have been shown to be highly successful in the treatment of angiotensin II-dependent hypertension (Ramahi, Postgrad. Med 2001; 109:115-122). This recently developed class of angiotensin II receptor blockers (ARBs) appear to be as effective as angiotensin-converting enzyme (ACE) inhibitors in delaying the progression of renal injury in animal models of diabetes (Barnett, Blood Press 2001; 10 Suppl 1:21-26). They act by selectively blocking the binding of angiotensin II to AGTR1 and may therefore offer a more complete blockade of the renin-angiotensin system than ACE inhibitors, which inhibit the conversion of angiotensin I to angiotensin II.
Unlike the angiotensin converting enzyme (ACE) inhibitors, these new drugs block the effects of angiotensin II regardless of whether it is produced systemically in the circulation or locally via ACE- or non-ACE-dependent pathways in tissues.
With the AGTR1 receptor blocked, angiotensin II is available to activate AGTR2, which mediates several potentially beneficial effects in the cardiovascular system, including vasodilation, antiproliferation, and apoptosis (Siragy, Am J Cardiol. 1999; 84:3S-8S). ARBs control a number of angiotensin II effects that are relevant to the pathophysiology of cardiovascular disease, including vasoconstriction, renal sodium reabsorption, aldosterone and vasopressin secretion, sympathetic activation, and vascular and cardiac hyperplasia and hypertrophy. Thus, ARBs provide a highly selective approach for regulating the effects of angiotensin II (Siragy, supra).
Most notable among ARBs is losartan, which has been found to be an effective anti-hypertension drug as it has an active metabolite that prolongs its duration of action. Other ARBs include valsartan, eprosartan, irbesartan, telmisartan, candesartan, and many others under investigation. Candesartan cilexetil requires conversion to an active form after administration. Telmisartan has the longest duration of action, with a terminal elimination half-life of around 24 hours in comparison with 11-15 hours for irbesartan, the agent with the next longest half-life (Burnier and Maillard, Blood Press 2001; 10 Suppl 1:6-11).
Therapy with any of the above drugs controls blood pressure in 40 to 50% of patients with mild to moderate hypertension. Tolerability has been reported to be very good, and ARBs would be a drug of choice in otherwise well-controlled hypertensive patients treated with angiotensin-converting enzyme inhibitors who developed cough or angioedema (Hernandez-Hernandez et al., J Hum Hypertens. 2000; 14 Suppl 1:S69-S72).
Angiotensin II has also been implicated in the development of cardiac hypertrophy, because ACE inhibitors and ARBs prevent or regress ventricular hypertrophy in animal models and in humans. Herzig et al. (Proc. Natl. Acad. Sci. U.S.A 1997; 94:7543-7548) studied AGTR1 promoter activity during cardiac hypertrophy, and discovered that AGTR1 expression is enhanced 160% in hypertrophied myocardium compared to normal myocardium, but that this effect could blocked by introducing mutations into either the AP-1 or GATA consensus binding sites within the AGTR1 promoter. These results suggest that the AP-1 and GATA consensus sites in the promoter regulate AGTR1 activity in cardiac muscle.
Several polymorphisms in the human AGTR1 gene have been discovered, some of which have been reported to be associated with hypertension. For example, Bonnardeaux et al. (Hypertension 1994; 24:63-69) identified an adenine or cytosine polymorphism (A1166C) located in the 3-prime untranslated region of the AGTR1 gene. This variant was present at a significantly elevated frequency in 206 Caucasian patients with essential hypertension. Wang et al. (Clin Genet 1997; 51:31-34) did a case-control study of the A1166C variant in a well-characterized group of 108 Caucasian hypertensive subjects with a strong family history (two affected parents) and early onset disease. The frequency of the A1166C allele in this subject group was 0.40 in hypertensives compared to 0.29 in normotensives. Further characterization of the A1629C polymorphism has shown it is significantly more frequent in women who develop pregnancy-induced hypertension as compared to healthy controls (Nalogowska-Glosnicka et al., Med Sci. Monit. 2000; 6:523-529). These data further support the notion that AGTR1 is an important target for the control of angiotensin II-dependent hypertension.
The angiotensin receptor 1 gene is located on chromosome 3q21-q25 and contains 1 exon that encodes a 359 amino acid protein. Two human AGTR1 subtypes have been identified, termed AGTR1A (FIG. 3) and AGTR1B, and recent evidence has indicated there may be as many four AGTR1 splice variants that are expressed in humans (Martin et al., Mol. Endocrinol. 2001; 15:281-293). AGTR1A and AGTR1B share substantial sequence homology and wide tissue distributions. AGTR1 seems to predominate in many tissues, but not in adrenal or anterior pituitary glands, and expression of the two types of receptors may be differentially regulated in the heart and the adrenals. This differential tissue distribution and regulation of AGTR1 subtypes may serve to modulate the biologic effects of angiotensin II (Ito et al., Proc. Natl. Acad. Sci. U.S.A 1995; 92:3521-3525). Reference sequences for the AGTR1 gene (Genaissance Reference No. 2506603; SEQ ID NO:1), coding sequence (GenBank Accession No:NMxe2x80x94000685.2), and protein are shown in FIGS. 1, 2 and 3, respectively.
Three additional known single nucleotide polymorphisms have been reported in the literature and correspond to thymine or cytosine at nucleotide position 1036 (NCBI SNP Database: Rs#5182), adenine or guanine at nucleotide position 1525 (NCBI SNP Database; Rs#5183), and thymine or guanine at nucleotide position 1613 (NCBI SNP Database; Rs#5185) in FIG. 1. Because of the potential for variation in the AGTR1 gene to affect the expression and function of the encoded protein, it would be useful to know whether additional polymorphisms exist in the AGTR1 gene, as well as how such polymorphisms are combined in different copies of the gene. Such information could be applied for studying the biological function of AGTR1 as well as in identifying drugs targeting this protein for the treatment of disorders related to its abnormal expression or function.
Accordingly, the inventors herein have discovered 4 novel polymorphic sites in the AGTR1 gene. These polymorphic sites (PS) correspond to the following nucleotide positions in FIG. 1: 104 (PS1), 348 (PS2), 1074 (PS4), and 1470 (PS5). The polymorphisms at these sites are thymine or adenine at PS1, guanine or thymine at PS2, thymine or cytosine at PS4, and cytosine or thymine at PS5. In addition, the inventors have determined the identity of the alleles at these sites, as well as at the previously identified sites at nucleotide positions 1036 (PS3), 1525 (PS6), and 1613 (PS7) in FIG. 1, in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic/Latino. From this information, the inventors deduced a set of haplotypes and haplotype pairs for PS 1-7 in the AGTR1 gene, which are shown below in Tables 5 and 4, respectively. Each of these AGTR1 haplotypes defines a naturally-occurring isoform (also referred to herein as an xe2x80x9cisogenexe2x80x9d) of the AGTR1 gene that exists in the human population. The frequency with which each haplotype and haplotype pair occurs within the total reference population and within each of the four major population groups included in the reference population was also determined.
Thus, in one embodiment, the invention provides a method, composition and kit for genotyping the AGTR1 gene in an individual. The genotyping method comprises identifying the nucleotide pair that is present at one or more polymorphic sites selected from the group consisting of PS1, PS2, PS4, and PS5 in both copies of the AGTR1 gene from the individual. A genotyping composition of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel AGTR1 polymorphic sites. A genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel AGTR1 polymorphic sites. In a preferred embodiment, the genotyping kit comprises a set of oligonucleotides designed to genotype each of PS 1-7. The genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 5 below or has one of the haplotype pairs in Table 4 below.
The invention also provides a method for haplotyping the AGTR1 gene in an individual. In one embodiment, the haplotyping method comprises determining, for one copy of the AGTR1 gene, the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PS1, PS2, PS4, and PS5. In another embodiment, the haplotyping method comprises determining whether one copy of the individual""s AGTR1 gene is defined by one of the AGTR1 haplotypes shown in Table 5, below, or a sub-haplotype thereof. In a preferred embodiment, the haplotyping method comprises determining whether both copies of the individual""s AGTR1 gene are defined by one of the AGTR1 haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof. The method for establishing the AGTR1 haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drugs for treating diseases associated with AGTR1 activity, e.g., hypertension.
For example, the haplotyping method can be used by the pharmaceutical research scientist to validate AGTR1 as a candidate target for treating a specific condition or disease predicted to be associated with AGTR1 activity. Determining for a particular population the frequency of one or more of the individual AGTR1 haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue AGTR1 as a target for treating the specific disease of interest. In particular, if variable AGTR1 activity is associated with the disease, then one or more AGTR1 haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls. Conversely, if each of the observed AGTR1 haplotypes are of similar frequencies in the disease and control groups, then it may be inferred that variable AGTR1 activity has little, if any, involvement with that disease. In either case, the pharmaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any AGTR1 haplotype or haplotype pair, apply the information derived from detecting AGTR1 haplotypes in an individual to decide whether modulating AGTR1 activity would be useful in treating the disease.
The claimed invention is also useful in screening for compounds targeting AGTR1 to treat a specific condition or disease predicted to be associated with AGTR1 activity. For example, detecting which of the AGTR1 haplotypes or haplotype pairs disclosed herein are present in individual members of a population with the specific disease of interest enables the pharmaceutical scientist to screen for a compound(s) that displays the highest desired agonist or antagonist activity for each of the most frequent AGTR1 isoforms present in the disease population. Thus, without requiring any a priori knowledge of the phenotypic effect of any particular AGTR1 haplotype or haplotype pair, the claimed haplotyping method provides the scientist with a tool to identify lead compounds that are more likely to show efficacy in clinical trials.
The method for haplotyping the AGTR1 gene in an individual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with AGTR1 activity. For example, instead of randomly assigning patients with the disease of interest to the treatment or control group as is typically done now, determining which of the AGTR1 haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute AGTR1 haplotypes and/or haplotype pairs evenly to treatment and control groups, thereby reducing the potential for bias in the results that could be introduced by a larger frequency of an AGTR1 haplotype or haplotype pair that had a previously unknown association with response to the drug being studied in the trial. Thus, by practicing the claimed invention, the scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any AGTR1 haplotype or haplotype pair.
In another embodiment, the invention provides a method for identifying an association between a trait and an AGTR1 genotype, haplotype, or haplotype pair for one or more of the novel polymorphic sites described herein. The method comprises comparing the frequency of the AGTR1 genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the AGTR1 genotype or haplotype in a reference population. A higher frequency of the AGTR1 genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the AGTR1 genotype, haplotype, or haplotype pair. In preferred embodiments, the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug. In a particularly preferred embodiment, the AGTR1 haplotype is selected from the haplotypes shown in Table 5, or a sub-haplotype thereof. Such methods have applicability in developing diagnostic tests and therapeutic treatments for hypertension.
In yet another embodiment, the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the AGTR1 gene or a fragment thereof. The reference sequence comprises SEQ ID NO:1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of adenine at PS1, thymine at PS2, cytosine at PS4, and thymine at PS5. In a preferred embodiment, the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of cytosine at PS3, guanine at PS6, and guanine at PS7.
A particularly preferred polymorphic variant is an isogene of the AGTR1 gene. An AGTR1 isogene of the invention comprises thymine or adenine at PS1, guanine or thymine at PS2, thymine or cytosine at PS3, thymine or cytosine at PS4, cytosine or thymine at PS5, adenine or guanine at PS6 and thymine or guanine at PS7. The invention also provides a collection of AGTR1 isogenes, referred to herein as an AGTR1 genome anthology.
In another embodiment, the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for an AGTR1 cDNA or a fragment thereof. The reference sequence comprises SEQ ID NO:2 (FIG. 2) and the polymorphic cDNA comprises at least one polymorphism selected from the group consisting of cytosine at a position corresponding to nucleotide 611 and thymine at a position corresponding to nucleotide 1007. In a preferred embodiment, the polymorphic variant comprises one or more additional polymorphisms selected from the group consisting of cytosine at a position corresponding to nucleotide 573 and guanine at a position corresponding to nucleotide 1062. A particularly preferred polymorphic cDNA variant comprises the coding sequence of an AGTR1 isogene defined by haplotypes 1-8 and 10.
Polynucleotides complementary to these AGTR1 genomic and cDNA variants are also provided by the invention. It is believed that polymorphic variants of the AGTR1 gene will be useful in studying the expression and function of AGTR1, and in expressing AGTR1 protein for use in screening for candidate drugs to treat diseases related to AGTR1 activity.
In other embodiments, the invention provides a recombinant expression vector comprising one of the polymorphic genomic variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector. The recombinant vector and host cell may be used to express AGTR1 for protein structure analysis and drug binding studies.
In yet another embodiment, the invention provides a polypeptide comprising a polymorphic variant of a reference amino acid sequence for the AGTR1 protein. The reference amino acid sequence comprises SEQ ID NO:3 (FIG. 3) and the polymorphic variant comprises at least one variant amino acid selected from the group consisting of serine at a position corresponding to amino acid position 204 and methionine at a position corresponding to amino acid position 336. A polymorphic variant of AGTR1 is useful in studying the effect of the variation on the biological activity of AGTR1 as well as on the binding affinity of candidate drugs targeting AGTR1 for the treatment of hypertension.
The present invention also provides antibodies that recognize and bind to the above polymorphic AGTR1 protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods.
The present invention also provides nonhuman transgenic animals comprising one of the AGTR1 polymorphic genomic variants described herein and methods for producing such animals. The transgenic animals are useful for studying expression of the AGTR1 isogenes in vivo, for in vivo screening and testing of drugs targeted against AGTR1 protein, and for testing the efficacy of therapeutic agents and compounds for hypertension in a biological system.
The present invention also provides a computer system for storing and displaying polymorphism data determined for the AGTR1 gene. The computer system comprises a computer processing unit; a display; and a database containing the polymorphism data. The polymorphism data includes the polymorphisms, the genotypes and the haplotypes identified for the AGTR1 gene in a reference population. In a preferred embodiment, the computer system is capable of producing a display showing AGTR1 haplotypes organized according to their evolutionary relationships.